Synthesis and structure-activity relationships of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone derivatives as potential agents against A549 lung cancer cells

Article abstract of DOI:10.1016/j.ejmech.2008.01.021

A series of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone derivatives were synthesized and the effects of all the compounds on A549 cell growth were investigated. The results showed that all compounds had almost inhibitory effects on the growth of A549 cells. The study on structure-activity relationships and prediction of lipophilicities of compounds showed that compounds with Log P values in the range of 4.12-6.80 had inhibitory effects on the growth of A549 cells, and among of them the hydrazone derived from salicylaldehyde had much more inhibitory effects.

Full text of DOI:10.1016/j.ejmech.2008.01.021

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 2347e2353
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and structureeactivity relationships of novel
1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone
derivatives as potential agents against A549 lung cancer cells
b
Yong Xia a, Chuan-Dong Fan b, Bao-Xiang Zhao a, , Jing Zhao ,
*
*
b,
Dong-Soo Shin c, , Jun-Ying Miao
*
a Institute of Organic Chemistry, School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China
b Institute of Developmental Biology, School of Life Science, Shandong University, Jinan 250100, China
c Department of Chemistry, Changwon National University, Changwon 641-773, South Korea
Received 12 September 2007; received in revised form 16 December 2007; accepted 10 January 2008
Available online 31 January 2008
Abstract
A series of novel 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone derivatives were synthesized and the effects of all the compounds
on A549 cell growth were investigated. The results showed that all compounds had almost inhibitory effects on the growth of A549 cells. The study on
structureeactivity relationships and prediction of lipophilicities of compounds showed that compounds with Log P values in the range of 4.12e6.80
had inhibitory effects on the growth of A549 cells, and among of them the hydrazone derived from salicylaldehyde had much more inhibitory effects.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Arylpyrazole carbohydrazide; Hydrazone; X-ray crystallography; A549 cells; Growth inhibitory; Structureeactivity relationship
1. Introduction
and the effects of 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohy-
drazide derivatives on A549 cell growth [14]. The study
on structureeactivity relationships and prediction of
lipophilicities of compounds showed that compounds with
Log P values in the range of 3.12e4.94 had more inhibitory ef-
fects on the growth of A549 cells. With the aim of obtaining
new anticancer compounds we synthesized a series of novel
1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydrazone
derivatives and evaluated effects of these compounds on
A549 cell growth inhibitory.
A number of hydrazideehydrazone derivatives have been
claimed to possess interesting bioactivity such as antibacteriale
antifungal [1], anticonvulsant [2], antiinflammatory [3], anti-
malarial [4], analgesic [5,6], antiplatelets [7], antituberculosis
[8] and anticancer activities [9]. Aroylhydrazideehydrazones
containing hetero-ring such as pyridine [3,10], indole [11],
1,2,4-oxadiazole [5], 1,2,3-triazole [6] and imidazo[2,1-
b][1,3,4]thiadiazole ring [8] have attracted special attention.
A few of pyrazole carbohydrazide hydrazone derivatives
have also been reported [12,13]. However, there has been no re-
port in the literature on the synthesis and biological evaluation
of 1-arylmethyl-3-aryl-1H-pyrazole-5-carbohydrazide hydra-
zone derivatives. In a previous paper we described the synthesis
2. Results and discussion
2.1. Chemistry
The synthesis of 1-arylmethyl-3-aryl-1H-pyrazole-5-carbo-
hydrazide hydrazone (II) has been accomplished as outlined in
Scheme 1 starting from 1-arylmethyl-3-aryl-1H-pyrazole-5-
carbohydrazide (I) that can be synthesized as described in
* Corresponding authors. Tel.: þ86 531 88366425; fax: þ86 531 88564464
(B.-X.Z.).
E-mail addresses: sduzhao@hotmail.com (B.-X. Zhao), miaojy@sdu.edu.
cn (J.-Y. Miao).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.021

2348
Y. Xia et al. / European Journal of Medicinal Chemistry 43 (2008) 2347e2353
R2
X
R2
X
ArCHO/EtOH
H
H
C
H
N
Ar
N
N
N
N
N
Reflux/2-14 h
NH2
N
C
C
O
O
R1
R1
1-20
II
I
R1 = H, Cl, OMe; R2 = H, Cl, t-Bu; X = C,N
HO
O
OMe
Ar =
O
O
d
b
c
a
Scheme 1. Synthesis of compounds 1e20.
our previous paper [14]. For example, (E )-N0-(benzo[d][1,3]-
dioxolo-5-ylmethylene)-1-benzyl-3-(4-chlorophenyl)-1H-pyr-
azole-5-carbohydrazide (1) was synthesized in 82% yield by
the reaction of 1-benzyl-3-(4-chlorophenyl)-1H-pyrazole-5-
carbohydrazide with piperonal in ethanol over a 4 h reflux
period. The structures of hydrazone derivatives II were deter-
mined by IR, 1H NMR and Mass spectroscopy. Thus, for
example 1, obtained as white crystal, gave an [M þ H]-ion
peak at m/z 459.5 in the ESI-MS, in accord with the molecular
formula C25H19ClN4O3. In the IR spectra, the carbonyl group
absorptions in hydrazide moiety and NH bands in CONH were
observed in the 1651 cmꢀ1 and 3203e2892 cmꢀ1 regions, re-
accepted as a reliable way to measure the cell proliferation
rate, and conversely when metabolic events lead to apoptosis
or necrosis. The data obtained by MTT assay showed that
compounds II had inhibitory effects on the growth of A549
cells in dosage- and time-dependent manners except com-
pounds 1, 5, 7, 9 and 11 as indicated by the results in
Fig. 2. As typically shown in Fig. 3, exposure of cells to com-
pounds 4, 8 and 12 at 10 mM for 24 h resulted in cell viability
decrease from 100% to 69, 61 and 80%, respectively.
1
spectively. The H NMR spectra indicated the chemical shift
of the NH at d ¼ 11.18 ppm in the form of singlet peak. The
chemical shift of the N]CH appeared at d ¼ 8.23 ppm in
the form of singlet peak. Two ortho-aromatic protons signals
in 4-chlorobenzene moiety appeared at the range of d ¼ 7.39
and 7.78 ppm as doublet peaks (J ¼ 8.4 Hz). Two ortho-aro-
matic protons signals in piperonyl moiety appeared at the
range of d ¼ 6.82 and 7.09 ppm as doublet peaks
(J ¼ 8.1 Hz). Three singlet signals appeared at d ¼ 5.85, 6.02
and 7.17 ppm are consistent with methylene protons in benzyl,
piperonyl groups and pyrazole moiety, respectively. Further-
more, the structure of (E )-N0-(2-hydroxybenzylidene)-1-(4-
tert-butylbenzyl)-3-phenyl-1H-pyrazole-5-carbohydrazide 16
was confirmed by the X-ray diffraction as shown in Fig. 1.
2.2. Evaluation of biological activity
2.2.1. Effects of the compounds on the viability of A549 lung
cancer cells
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) cell proliferation assay has been widely
Fig. 1. X-ray crystal structures of compound 16. Displacement ellipsoids are
drawn at 50% probability level.

Y. Xia et al. / European Journal of Medicinal Chemistry 43 (2008) 2347e2353
2349
and 15e20 was conducted. The exposure of A549 cells to
compounds 4, 8, 12 and 15e20 for the duration of 24, 48,
and 96 h resulted in significant increase in the cell prolifera-
tion inhibitory percentage.
2.2.2. Structureeactivity relationships
Growth inhibitory properties (IC50) are listed. The data
suggested that compounds could almost inhibit the cell
growth obviously in the concentrations of 0.5e20 mM after
96 h of the treatment except compounds 1, 5, 7, 9 and 11.
In our previous paper, we reported that 1-arylmethyl-3-aryl-
1H-pyrazole-5-carbohydrazide derivatives, which are the
starting material in this study, could inhibit the A549 cell
growth obviously in the concentrations of 20e80 mM after
48 h of the treatment and the cytotoxic potency was highly
dependent on the substitution types and patterns on the aryl
ring, for example, replacing the hydrogen at the 4-position
of 1-aryl ring with a bulkier tert-butyl group resulted in a sig-
nificant activity increasing. In present study, we observed that
the nature and the position of substituent on the molecule
improve biological functions. Regarding the Ar substituted-
hydrazone structure variants a, b, c and d, the antitumor
activity of these compounds appears to be closely related to
Fig. 2. Growth inhibitory properties IC50 (mM) for the compounds at 96 h.
Continuing the exposure for 48 h resulted in cell viability de-
crease from 100% to 41, 32 and 45%, respectively. When the
exposure continued on to 96 h, compared with the control
group, the cell viability reduced more significantly from
100% to 6, 7 and 10% ( p < 0.01). Further, exposure of cells
to compounds 4, 8 and 12 at 20 mM for 48 h, the cell viability
reduced more significantly from 100% to 38, 18 and 34%, re-
spectively. The compounds 15e20 showed the similar dosage-
and time-dependent manners as shown in Fig. 3B and C.
Further investigation of the concentration- and time-depen-
dent viability of A549 cells exposed to compounds 4, 8, 12
Fig. 3. (A) Effects of the compounds 4, 8 and 12 on the viability of A549 lung cancer cells. (B) Effects of the compounds 15e17 on the viability of A549 lung
cancer cells. (C) Effects of the compounds 18e20 on the viability of A549 lung cancer cells.

2350
Y. Xia et al. / European Journal of Medicinal Chemistry 43 (2008) 2347e2353
the nature of the Ar group. Compounds 4, 8, 12 and 15e20
with b group were proved to be the most active member
with a unique antitumor potency against A549 cell lines. Fur-
thermore, replacement of the benzyl group with a t-butylben-
zyl moiety as in 16, 18 and 20, resulted in a higher growth
inhibitory effect.
When Ar group was a and c, the compounds were difficult
to dissolve in RPMI 1640 medium so that the activity of these
compounds could not be examined in suitable concentration.
Here, it should be pointed out that the solubility data (Log S )
obtained by calculating were not concordant with the solubil-
ity in RPMI 1640 medium examined by the experimental
method as shown in Table 1. When Ar was d, the growth in-
hibitory properties of the compounds were inferior to the com-
pounds with b group, although they had a good solubility in
RPMI 1640 medium.
and non-polar phases, and it has been demonstrated to be an
indispensable tool in predicting the transport and activity of
drugs [15e18]. Prediction of lipophilicity (Log P) and aque-
ous solubility (Log S ) of compounds 1e20 was calculated us-
ing ALOGPS 2.1 software [19,20]. Assuming that the issue of
penetration is even more crucial for compound’s activity
against cell, our results demonstrated that simply increasing
the lipophilic character of compounds increased the activity,
as shown with Log P values of the synthesized compounds
hydrazone (4.12e6.80) (Table 1) were much more than the
parent compound hydrazide (2.52e4.94).
3. Conclusion
The biological evaluation of 1-arylmethyl-3-aryl-1H-pyra-
zole-5-carbohydrazide hydrazone derivatives 1e20 on A549
lung cancer cell growth showed that the hydrazone com-
pounds derived from salicylaldehyde inhibited much more
proliferation of A549 cell. It was suggested that the potency
of these compounds against A549 lung cancer cell not only
related with the lipophilicity of the compound but also
depended on the capacity of compound chelating metal ions
because it was recognized that metal ions play an important
role in the cell growth. As potent chelators of ions that effec-
tively inhibit tumor cell growth, the novel compounds cer-
tainly deserve further careful investigation in terms of their
possible mechanism. Currently, investigations are underway
to elucidate the mechanism and the results will be published
in due course.
These results revealed the crucial role of the o-hydroxyben-
zene moiety in the hydrazone cytotoxic activities. It should be
raised from the possibility of compounds chelating with some
ions in cell.
Compared the growth inhibitory properties (IC50) of com-
pounds I and II, it was obvious that compounds II were more
effective. For example, after 48 h of the treatment, the IC50
for compound 18 and 20 was 3.33 and 4.80 mM, however, the
IC50 for corresponding parent compound, 1-(4-tert-butylben-
zyl)-3-(4-chlorophenyl)-1H-pyrazole-5-carbohydrazide and
1-(4-tert-butylbenzyl)-3-(4-methoxyphenyl)-1H-pyrazole-5-
carbohydrazide were 18.52 and 23.86 mM, respectively.
The lipophilicity of the compounds is well known to play
an important role in the penetration of these compounds into
cells. The partition coefficient Log P is a parameter which de-
scribes the manner in which a drug partitions between polar
4. Experimental
Thin-layer chromatography (TLC) was carried out on silica
gel 60 F254 plates (Merck KGaA). 1H NMR spectra were
recorded on a Bruker Avance 300 (300 MHz) spectrometer,
using CDCl3 or DMSO as solvents and tetramethylsilane
(TMS) as internal standard. Melting points were determined
on an XD-4 digital micro melting point apparatus and are un-
corrected. IR spectra were recorded with an IR spectropho-
tometer Avtar 370 FT-IR (Termo Nicolet). MS spectra were
recorded on a Trace DSQ mass spectrograph. Log P and
Log S were calculated using ALOGPS 2.1 software.
Table 1
The IC50, Log P, Log S and solubility in 1640 of the compounds 1e20
Compound R1
R2
X
Ar Smax in Log S Log P IC50 (mM)
1640
(mM)
(mM) (mM)
48 h 96 h
1
Cl
Cl
H
H
C
C
N
N
N
N
N
N
N
N
N
N
N
N
C
C
C
C
C
C
a
d
a
b
c
8.0
12.5
17.0
45.0
15.0
50.0
2.0
4.92 4.96
49.80 4.76
15.83 4.35
12.13 4.86
5.36 4.58
72.59 4.12
8.48 4.79
6.80 5.38
3.41 5.17
60.08 4.65
15.04 4.36
13.62 4.87
5.67 4.80
74.54 4.20
10.70 4.88
1.08 6.25
4.64 5.48
0.60 6.80
8.86 4.86
1.06 6.25
Nd
Nd
Nd
Nd
8.78
12.16
2
H
3
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
4
H
5.83 0.44
Nd Nd
26.13 19.38
5
H
6
H
d
a
b
c
7
Cl
Cl
Cl
Cl
Nd Nd
3.68 0.50
4.1. General procedure for the synthesis of 1-arylmethyl-
3-aryl-1H-pyrazole-5-carbohydrazide hydrazone
derivatives (1e20)
8
22.5
3.3
9
Nd
18.65 9.82
Nd
10
11
12
13
14
15
16
17
18
19
20
d
a
b
c
70.0
7.5
OMe Cl
OMe Cl
OMe Cl
OMe Cl
Nd Nd
8.21 0.57
To a stirred solution of 1 mmol of derivatives I in 10 ml of
ethanol, there was added an equimolar amount of the appropri-
ate aldehyde derivative. The reaction mixture was maintained
under reflux for 2e14 h, until TLC indicated the end of reac-
tion. After this time, the reaction mixture stood over night and
the solid formed was collected by filtration and washed with
ethanol and recrystallized from ethanol to afford crystals. As
a result of this process the compounds II (1e20) were pre-
pared in yield of 82e98%.
70.0
17.5
30.0
44.0
47.0
25.0
36.0
23.0
22.0
Nd
Nd
18.91
15.79
d
b
b
b
b
b
b
H
H
10.76 0.65
6.24 0.56
4.87 0.68
3.33 0.43
7.69 0.84
4.80 0.55
H
Cl
t-Bu
H
Cl
OMe
t-Bu
H
OMe t-Bu
Nd: not determined.

Y. Xia et al. / European Journal of Medicinal Chemistry 43 (2008) 2347e2353
2351
4.1.1. (E )-N0-(benzo[d][1,3]dioxolo-5-ylmethylene)-
4.1.6. (E )-1-((6-chloropyridin-3-yl)methyl)-N0-
(furan-2-ylmethylene)-3-phenyl-1H-pyrazole-
5-carbo-hydrazide (6)
1-benzyl-3-(4-chlorophenyl)-1H-pyrazole-
5-carbohydrazide (1)
White solid, yield 82%, mp 233e236 ꢁC; IR (KBr) n:
White solid, yield 98%, mp 210e212 ꢁC; IR (KBr) n:
1
1
3211e2892 (NH), 1651 (C]O) cmꢀ1; H NMR (300 MHz,
3204e3049 (NH), 1649 (C]O) cmꢀ1; H NMR (300 MHz,
CDCl3 þ DMSO) d: 5.85 (s, 2H, CH2), 6.02 (s, 2H, Oe
CH2), 6.82 (d, J ¼ 8.1 Hz, 1H, ArH), 7.09 (d, J ¼ 8.1 Hz,
1H, ArH), 7.17 (s, 1H, 4-H), 7.26e7.35 (m, 3H, ArH), 7.39
(d, J ¼ 8.4 Hz, 2H, ArH), 7.42e7.46 (m, 3H, ArH), 7.78 (d,
J ¼ 8.4 Hz, 2H, ArH), 8.23 (s, 1H, ]CH), 11.18 (s, 1H,
NH); ESI-MS: 459.5 (M þ H)þ.
CDCl3 þ DMSO) d: 5.86 (s, 2H, CH2), 6.51 (s, 1H, FuH),
6.87 (s, 1H, FuH), 7.24 (s, 1H, 4-H), 7.27e7.43 (m, 5H,
ArH, PyH, FuH), 7.55e7.83 (m, 3H, ArH, PyH), 8.31 (s,
1H, ]CH), 8.50 (s, 1H, PyH), 11.57 (s, 1H, NH); ESI-MS:
406.6 (M þ H)þ.
4.1.7. (E )-N0-(benzo[d][1,3]dioxolo-5-ylmethylene)-3-
(4-chlorophenyl)-1-((6-chloropyridin-3-yl)methyl)-1H-
pyrazole-5-carbohydrazide (7)
4.1.2. (E )-1-benzyl-3-(4-chlorophenyl)-N0-(furan-
2-ylmethylene)-1H-pyrazole-5-carbohydrazide (2)
White solid, yield 92%, mp 249e251 ꢁC; IR (KBr) n:
White solid, yield 94%, mp 256e258 ꢁC; IR (KBr) n:
1
3165e2955 (NH), 1660 (C]O) cmꢀ1; H NMR (400 MHz,
1
3198e3062 (NH), 1651 (C]O) cmꢀ1; H NMR (400 MHz,
DMSO) d: 5.80 (s, 2H, CH2), 6.10 (s, 2H, OeCH2), 7.01 (d,
J ¼ 8.0 Hz, 1H, ArH), 7.20 (d, J ¼ 8.0 Hz, 1H, ArH), 7.30
(s, 1H, ArH), 7.47 (s, 1H, 4-H), 7.50 (d, J ¼ 8.4 Hz, 1H,
PyH), 7.54 (d, J ¼ 8.4 Hz, 2H, ArH), 7.74 (dd, J ¼ 2.1 Hz,
8.4 Hz, 1H, PyH), 7.82 (d, J ¼ 8.4 Hz, 2H, ArH), 8.32 (s,
1H, ]CH), 8.37 (d, J ¼ 2.1 Hz, 1H, PyH), 11.91 (s, 1H,
NH); ESI-MS: 494.3 (M þ H)þ.
CDCl3 þ DMSO) d: 5.84 (s, 2H, CH2), 6.50e6.51 (m, 1H,
FuH), 6.84 (d, J ¼ 2.1 Hz, 1H, FuH), 7.20 (s, 1H, 4-H),
7.22e7.29 (m, 3H, ArH), 7.35e7.42 (m, 4H, ArH), 7.54 (s,
1H, FuH), 7.77 (d, J ¼ 8.4 Hz, 2H, ArH), 8.29 (s, 1H,
]CH), 11.51 (s, 1H, NH); ESI-MS: 405.5 (M þ H)þ.
4.1.3. (E )-N0-(benzo[d][1,3]dioxolo-5-ylmethylene)-1-
((6-chloropyridin-3-yl)methyl)-3-phenyl-1H-pyrazole-
5-carbohydrazide (3)
4.1.8. (E )-3-(4-chlorophenyl)-1-((6-chloropyridin-
3-yl)methyl)-N0-(2-hydroxybenzylidene)-1H-
pyrazole-5-carbohydrazide (8)
Yellow solid, yield 96%, mp 231e233 ꢁC; IR (KBr) n: 3615
(OH), 3216e3059 (NH), 1677 (C]O) cmꢀ1 1H NMR
;
(400 MHz, CDCl3 þ DMSO) d: 5.79 (s, 2H, CH2), 6.82e
6.88 (m, 2H, ArH), 7.20e7.28 (m, 2H, ArH), 7.33e7.36 (m,
4H, ArH, PyH, 4-H), 7.71e7.73 (m, 3H, ArH, PyH), 8.36 (s,
1H, ]CH), 8.49 (s, 1H, PyH), 11.10 (s, 1H, OH), 11.99 (s,
1H, NH); ESI-MS: 466.4 (M þ H)þ.
Yellow solid, yield 87%, mp 213e215 ꢁC; IR (KBr) n:
1
3173e2952 (NH), 1662 (C]O) cmꢀ1; H NMR (400 MHz,
DMSO) d: 5.81 (s, 2H, CH2), 6.10 (s, 2H, OeCH2), 7.01 (d,
J ¼ 8.0 Hz, 1H, ArH), 7.20 (d, J ¼ 8.0 Hz, 1H, ArH), 7.30
(s, 1H, 4-H), 7.37e7.51 (m, 5H, ArH), 7.73 (dd, J ¼ 2.1 Hz,
8.4 Hz, 1H, PyH), 7.81 (d, J ¼ 7.6 Hz, 2H, ArH), 8.33 (s,
1H, ]CH), 8.37 (d, J ¼ 2.1 Hz, 1H, PyH), 11.90 (s, 1H,
NH); ESI-MS: 460.5 (M þ H)þ.
4.1.9. (E )-3-(4-chlorophenyl)-1-((6-chloropyridin-
3-yl)methyl)-N0-(4-methoxybenzylidene)-1H-
pyrazole-5-carbohydrazide (9)
4.1.4. (E )-1-((6-chloropyridin-3-yl)methyl)-N0-
(2-hydroxybenzylidene)-3-phenyl-1H-
Yellow solid, yield 94%, mp 235e237 ꢁC; IR (KBr) n:
pyrazole-5-carbohydrazide (4)
3173e2961 (NH), 1653 (C]O), 1267 (OeC) cmꢀ1 1H
;
Yellow solid, yield 89%, mp 99e100 ꢁC; IR (KBr) n:
1
3429e2959 (NH), 1664 (C]O) cmꢀ1; H NMR (300 MHz,
NMR (400 MHz, DMSO) d: 3.82 (s, 3H, OMe), 5.81 (s, 2H,
CH2), 7.04 (d, J ¼ 8.4 Hz, 2H, ArH), 7.47 (s, 1H, 4-H), 7.50
(d, J ¼ 8.4 Hz, 1H, PyH), 7.53 (d, J ¼ 8.4 Hz, 2H, ArH),
7.69 (d, J ¼ 8.4 Hz, 2H, ArH), 7.74 (dd, J ¼ 2.2 Hz, 8.4 Hz,
1H, PyH), 7.82 (d, J ¼ 8.4 Hz, 2H, ArH), 8.35 (s, 1H,
]CH), 8.38 (d, J ¼ 2.2 Hz, 1H, PyH), 11.88 (s, 1H, NH);
ESI-MS: 480.4 (M þ H)þ.
CDCl3) d: 5.78 (s, 2H, CH2), 6.89e7.02 (m, 3H, 4-H, ArH),
7.19e7.43 (m, 7H, ArH), 7.73e7.74 (m, 3H, ArH, PyH,
NH), 8.38 (s, 1H,]CH), 8.43 (s, 1H, PyH), 9.50 (s, 1H,
OH); ESI-MS: 432.5 (M þ H)þ.
4.1.5. (E )-1-((6-chloropyridin-3-yl)methyl)-N0-
(4-methoxybenzylidene)-3-phenyl-1H-
4.1.10. (E )-3-(4-chlorophenyl)-1-((6-chloropyridin-
3-yl)methyl)-N0-(furan-2-ylmethylene)-1H-
pyrazole-5-carbohydrazide (5)
White solid, yield 98%, mp 213e215 ꢁC; IR (KBr) n:
pyrazole-5-carbohydrazide (10)
1
3441e2838 (NH), 1651 (C]O) cmꢀ1; H NMR (300 MHz,
White solid, yield 81%, mp 178e180 ꢁC; IR (KBr) n:
1
CDCl3) d: 3.85 (s, 3H, OMe), 5.83 (s, 2H, CH2), 6.94e7.02
(m, 3H, ArH), 7.23 (s, 1H, 4-H), 7.35e7.42 (m, 3H, ArH,
PyH), 7.59e7.79 (m, 5H, ArH, PyH), 8.14 (s, 1H, ]CH),
8.50 (s, 1H, PyH), 9.21 (s, 1H, NH); ESI-MS: 446.5
(M þ H)þ.
3419e3051 (NH), 1654 (C]O) cmꢀ1; H NMR (400 MHz,
DMSO) d: 5.80 (s, 2H, CH2), 6.66 (s, 1H, FuH), 6.99 (d,
J ¼ 3.1 Hz, 1H, FuH), 7.45 (s, 1H, 4-H), 7.50 (d, J ¼ 8.6 Hz,
1H, PyH), 7.53 (d, J ¼ 8.4 Hz, 2H, ArH), 7.73 (d,
J ¼ 8.6 Hz, 1H, PyH), 7.83 (d, J ¼ 8.4 Hz, 2H, ArH), 7.88

2352
Y. Xia et al. / European Journal of Medicinal Chemistry 43 (2008) 2347e2353
(s, 1H, FuH), 8.28 (s, 1H, ]CH), 8.37 (s, 1H, PyH), 11.95 (s,
1H, NH); ESI-MS: 440.5 (M þ H)þ.
CDCl3 þ DMSO) d: 5.74 (s, 2H, CH2), 6.77 (t, J ¼ 8.0 Hz,
1H, ArH), 6.87 (d, J ¼ 8.4 Hz, 1H, ArH), 7.10e7.32 (m,
11H, 4-H, ArH), 7.72 (d, J ¼ 8.0 Hz, 2H, ArH), 8.34 (s, 1H,
]CH), 11.23 (s, 1H, OH), 11.52 (s, 1H, NH); ESI-MS:
397.5 (M þ H)þ.
4.1.11. (E )-N0-(benzo[d][1,3]dioxolo-5-ylmethylene)-1-
((6-chloropyridin-3-yl)methyl)-3-(4-methoxyphenyl)-
1H-pyrazole-5-carbohydrazide (11)
Yellow solid, yield 92%, mp 208e209 ꢁC; IR (KBr) n:
3176e2957 (NH), 1652 (C]O), 1260 (OeC) cmꢀ1 1H
;
NMR (400 MHz, DMSO) d: 3.79 (s, 3H, OMe), 5.79 (s, 2H,
CH2), 6.10 (s, 2H, OeCH2), 7.00 (d, J ¼ 8.0 Hz, 1H, ArH),
7.02 (d, J ¼ 8.4 Hz, 2H, ArH), 7.19 (d, J ¼ 8.0 Hz, 1H, ArH),
7.30 (s, 1H, 4-H), 7.37 (s, 1H, ArH), 7.50 (d, J ¼ 8.0 Hz, 1H,
PyH), 7.72 (dd, J ¼ 2.2 Hz, 8.4 Hz, 1H, PyH), 7.73 (d,
J ¼ 8.4 Hz, 2H, ArH), 8.32 (s, 1H ]CH), 8.36 (d,
J ¼ 2.2 Hz, 1H, PyH), 11.87 (s, 1H, NH); ESI-MS: 490.4 (M)þ.
4.1.16. (E )-N0-(2-hydroxybenzylidene)-1-(4-
tert-butylbenzyl)-3-phenyl-1H-pyrazole-
5-carbohydrazide (16)
White solid, yield 97%, mp 106e108 ꢁC; IR (KBr) n:
1
3480e2957 (NH), 1664 (C]O) cmꢀ1; H NMR (400 MHz,
CDCl3 þ DMSO) d: 1.19 (s, 9H, 3Me), 5.74 (s, 2H, CH2),
6.81e6.88 (m, 2H, ArH), 7.18e7.29 (m, 9H, ArH, 4-H,
OH), 7.34 (t, J ¼ 7.6 Hz, 2H, ArH), 7.75 (d, J ¼ 7.6 Hz, 2H,
ArH), 8.45 (s, 1H, ]CH), 11.86 (s, 1H, NH); ESI-MS:
453.6 (M þ H)þ.
4.1.12. (E )-1-((6-chloropyridin-3-yl)methyl)-N0-
(2-hydroxybenzylidene)-3-(4-methoxyphenyl)-
1H-pyrazole-5-carbohydrazide (12)
4.1.17. (E )-N0-(2-hydroxybenzylidene)-1-benzyl-3-
(4-clorophenyl)-1H-pyrazole-5-carbohydrazide (17)
White solid, yield 98%, mp 208e209 ꢁC; IR (KBr) n:
Yellow solid, yield 98%, mp 100e102 ꢁC; IR (KBr) n: 3560
(OH), 3209e2833 (NH), 1680 (C]O) cmꢀ1
;
1H NMR
3234e3019 (NH), 1649 (C]O) cmꢀ1; H NMR (400 MHz,
1
(400 MHz, CDCl3 þ DMSO) d: 3.77 (s, 3H, OMe), 5.76 (s,
2H, CH2), 6.81e6.92 (m, 5H, ArH, OH), 7.20e7.22 (m, 4H,
ArH, PyH, 4-H), 7.66 (d, J ¼ 8.4 Hz, 2H, ArH), 7.68 (dd,
J ¼ 2.2 Hz, 8.4 Hz, 1H, PyH), 8.35 (d, J ¼ 2.2 Hz, 1H, PyH),
8.45 (s, 1H, ]CH), 11.85 (s, 1H, NH); ESI-MS: 462.4
(M þ H)þ.
CDCl3 þ DMSO) d: 5.77 (s, 2H, CH2), 6.80e6.90 (m, 2H,
ArH), 7.14e7.28 (m, 8H, ArH, 4-H,), 7.32 (d, J ¼ 8.4 Hz,
2H, ArH), 7.72 (d, J ¼ 8.4 Hz, 2H, ArH), 8.43 (s, 1H,
]CH), 11.21 (s, 1H, OH), 11.86 (s, 1H, NH); ESI-MS:
431.5 (M þ H)þ.
4.1.18. (E )-N0-(2-hydroxybenzylidene)-1-(4-
tert-butylbenzyl)-3-(4-chlorophenyl)-1H-
pyrazole-5-carbohydrazide (18)
4.1.13. (E )-1-((6-chloropyridin-3-yl)methyl)-N0-
(4-methoxybenzylidene)-3-(4-methoxyphenyl)-
1H-pyrazole-5-carbohydrazide (13)
White solid, yield 92%, mp 206e208 ꢁC; IR (KBr) n:
1
Yellow solid, yield 91%, mp 205e206 ꢁC; IR (KBr) n:
3173e2866 (NH), 1657 (C]O) cmꢀ1; H NMR (400 MHz,
3174e2837 (NH), 1654 (C]O), 1263 (OeC) cmꢀ1
;
1H
CDCl3 þ DMSO) d: 1.19 (s, 9H, 3Me), 5.73 (s, 2H, CH2),
6.81e6.89 (m, 2H, ArH), 7.18e7.29 (m, 7H, ArH, 4-H),
7.33 (d, J ¼ 8.4 Hz, 2H, ArH), 7.72 (d, J ¼ 8.4 Hz, 2H,
ArH), 8.44 (s, 1H ]CH), 11.20 (s, 1H, OH), 11.89 (s, 1H,
NH); ESI-MS: 487.5 (M þ H)þ.
NMR (400 MHz, DMSO) d: 3.79 (s, 3H, OMe), 3.82 (s, 3H,
OMe), 5.79 (s, 2H, CH2), 7.02 (J ¼ 8.4 Hz, 2H, ArH), 7.03
(d, J ¼ 8.4 Hz, 2H, ArH), 7.37 (s, 1H, 4-H), 7.49 (d,
J ¼ 8.4 Hz, 1H, PyH), 7.68 (d, J ¼ 8.4 Hz, 2H, ArH), 7.71e
7.75 (m, 3H, ArH, PyH), 8.35 (s, 1H ]CH), 8.37 (s, 1H,
PyH), 11.83 (s, 1H, NH); ESI-MS: 476.4 (M þ H)þ.
4.1.19. (E )-N0-(2-hydroxybenzylidene)-1-benzyl-3-
(4-methoxyphenyl)-1H-pyrazole-5-carbohydrazide (19)
White solid, yield 90%, mp 206e208 ꢁC; IR (KBr) n:
4.1.14. (E )-1-((6-chloropyridin-3-yl)methyl)-N0-(furan-
2-ylmethylene)-3-(4-methoxyphenyl)-1H-pyrazole-5-
carbohydrazide (14)
1
3201e2828 (NH), 1657 (C]O) cmꢀ1; H NMR (400 MHz,
CDCl3 þ DMSO) d: 3.76 (s, 3H, OMe), 5.76 (s, 2H, CH2),
6.81e6.89 (m, 4H, ArH), 7.11e7.26 (m, 8H, ArH, 4-H),
7.58 (s, 1H, OH), 7.67 (d, J ¼ 8.4 Hz, 2H, ArH), 8.44 (s,
1H, ]CH), 11.84 (s, 1H, NH); ESI-MS: 427.5 (M þ H)þ.
Yellow solid, yield 91%, mp 200e202 ꢁC; IR (KBr) n:
1
3211e2927 (NH), 1655 (C]O) cmꢀ1; H NMR (400 MHz,
DMSO) d: 3.79 (s, 3H, OMe), 5.78 (s, 2H, CH2), 6.64e6.66
(m, 1H, FuH), 6.98 (d, J ¼ 3.0 Hz, 1H, FuH), 7.02 (d,
J ¼ 8.4 Hz, 2H, ArH), 7.35 (s, 1H, 4-H), 7.50 (d, J ¼ 8.4 Hz,
1H, PyH), 7.72 (dd, J ¼ 2.0, 8.4 Hz, 1H, PyH), 7.74 (d,
J ¼ 8.4 Hz, 2H, ArH), 7.87 (s, 1H, FuH), 8.29 (s, 1H,
]CH), 8.36 (d, J ¼ 2.0 Hz, 1H, PyH), 11.91 (s, 1H, NH);
ESI-MS: 436.5 (M þ H)þ.
4.1.20. (E )-N0-(2-hydroxybenzylidene)-1-(4-
tert-butylbenzyl)-3-(4-methoxyphenyl)-1H-
pyrazole-5-carbohydrazide (20)
Yellow solid, yield 92%, mp 158e160 ꢁC; IR (KBr) n:
1
3523e2962 (NH), 1659 (C]O) cmꢀ1; H NMR (400 MHz,
CDCl3 þ DMSO) d: 1.18 (s, 9H, 3Me), 3.76 (s, 3H, OMe),
5.73 (s, 2H, CH2), 6.80e6.93 (m, 4H, ArH), 7.09 (s, 1H, 4-
H), 7.16e7.22 (m, 7H, ArH, OH), 7.67 (d, J ¼ 8.4 Hz, 2H,
ArH), 8.40 (s, 1H, ]CH), 11.67 (s, 1H, NH); ESI-MS:
483.5 (M þ H)þ.
4.1.15. (E )-N0-(2-hydroxybenzylidene)-1-benzyl-3-
phenyl-1H-pyrazole-5-carbohydrazide (15)
White solid, yield 82%, mp 234e236 ꢁC; IR (KBr) n:
1
3246e2931 (NH), 1660 (C]O) cmꢀ1; H NMR (400 MHz,

Y. Xia et al. / European Journal of Medicinal Chemistry 43 (2008) 2347e2353
2353
4.2. Biological activity assay
analyzed by t-test. Differences at p < 0.05 were considered
statistically significant.
4.2.1. Materials
RPMI 1640 was obtained from Gibco BRL Co. (Grand Island,
USA) andbovinecalf serumwasfromBeijingDingGuoBiotech-
nology Co. (China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide (MTT) was purchased from Amresco.
Acknowledgments
This study was supported by the Foundation of the Ministry
of Education (104112) and the Natural Science Foundation of
Shandong Province (Y2005B12).
4.2.2. Cell culture
References
A549 lung cancer cells were cultured in RPMI 1640 me-
dium, supplemented with 10% (v/v) newborn calf serum at
37 ꢁC in 5% CO2, and 95% air. The cells were routinely
seeded at the density of 3125/cm2 into 96-well plates or other
appropriate dishes containing the medium.
[1] C. Loncle, J.M. Brunel, N. Vidal, M. Dherbomez, Y. Letourneux, Eur.
J. Med. Chem. 39 (2004) 1067e1071.
¨
[2] Sx.G. Kuc¸ukguzel, A. Mazi, F. Sahin, S. Ozturk, J. Stables, Eur. J. Med.
¨ ¨
Chem. 38 (2003) 1005e1013.
¨
¨
[3] A.R. Todeschini, A.L.P. de Miranda, K.C.M. da Silva, S.C. Parrini,
E.J. Barreiro, Eur. J. Med. Chem. 33 (1998) 189e199.
[4] P. Melnyk, V. Leroux, C. Sergheraert, P. Grellier, Bioorg. Med. Chem.
Lett. 16 (2006) 31e35.
4.2.3. MTT assay for cell viability
The compounds were dissolved in DMSO. The final con-
centration of DMSO was below 0.1% in the culture medium
(v/v) (DMSO at these final concentrations did not affect the vi-
ability of the cells). Cells were seeded in 96-well plates and
treated with compounds 1e20 at 0.1e40 mM for 24, 48 and
96 h, respectively. The cell viability was determined by the
MTT assay following the procedure described by Price and
McMillan [21]. The light absorptions were measured at
570 nm using SpectraMAX 190 microplate spectrophotometer
(GMI Co., USA).
[5] L.F.C.C. Leite, M.N. Ramos, J.B.P. da Silva, A.L.P. Miranda,
C.A.M. Fraga, E.J. Barreiro, Farmaco 54 (1999) 747e757.
´
[6] P.C. Lima, L.M. Lima, K.C.M. da Silva, P.H.O. Leda, A.L.P. de Miranda,
C.A.M. Fraga, E.J. Barreiro, Eur. J. Med. Chem. 35 (2000) 187e203.
[7] A.C. Cunha, J.M. Figueiredo, J.L.M. Tributino, A.L.P. Miranda,
H.C. Castro, R.B. Zingali, C.A.M. Fraga, M.C.B.V. de Souza,
V.F. Ferreira, E.J. Barreiro, Bioorg. Med. Chem. 11 (2003)
2051e2059.
[8] K.K. Bedia, O. Elc¸in, U. Seda, K. Fatma, S. Nathaly, R. Sevim,
A. Dimoglo, Eur. J. Med. Chem. 41 (2006) 1253e1261.
[9] N. Terzioglu, A. Gursoy, Eur. J. Med. Chem. 38 (2003) 781e786.
´
¨
[10] N. Galic, B. Peric, B. Kojic-Prodic, Z. Cimerman, J. Mol. Struct. 559
(2001) 187e194.
´
´
´
4.2.4. Determination of solubility of compounds
in RPMI 1640 medium
¨
¨
[11] F.B. Kaynak, D. Ozturk, S. Ozbey, G. C¸ apan, J. Mol. Struct. 740 (2005)
¨
213e221.
The compounds were dissolved in supposed range of con-
centrations at an interval of 0.5 mM in RPMI 1640 medium,
supplemented with 10% (v/v) Bovine Calf Serum, at 37 ꢁC.
Then the medium was added into 24-well plates in which
had been seeded A549 cells at a density of 3125/cm2. The
plates were incubated at 37 ꢁC in 5% CO2, and 95% air, in
an incubator. The compound was considered to be dissolved
completely if there were no visible crystals observed under
Phase Contrast Microscope (Nikon, Japan) in the medium at
24 h. The maximum concentration at which the compound dis-
solved completely was considered the dissolvability of the
compound in the medium. We got the dissolvability of the
compounds from three independent experiments.
[12] Sh.A.F. Rostom, M.A. Shalaby, M.A. El-Demellawy, Eur. J. Med. Chem.
38 (2003) 959e974.
[13] A.M.R. Bernardino, A.O. Gomes, K.S. Charret, A.C.C. Freitas,
G.M.C. Machado, M.M. Canto-Cavalheiro, L.L. Leon, V.F. Amaral,
Eur. J. Med. Chem. 41 (2006) 80e87.
[14] Y. Xia, Z.W. Dong, B.X. Zhao, X. Ge, N. Meng, D.S. Shin, J.Y. Miao,
Bioorg. Med. Chem. 15 (2007) 6893e6899.
[15] F. Grande, F. Aiello, O.D. Grazia, A. Brizzi, A. Garofalo, N. Neamati,
Bioorg. Med. Chem. 15 (2007) 288e294.
[16] T. Yakaiah, B.P.V. Lingaiah, B. Narsaiah, B. Shireesha, B. Ashok Kumar,
S. Gururaj, T. Parthasarathy, B. Sridhar, Bioorg. Med. Chem. Lett. 17
(2007) 3445e3453.
[17] A. Kamal, M.N.A. Khan, K.S. Reddy, K. Rohini, Bioorg. Med. Chem. 15
(2007) 1004e1013.
[18] R.Y. Leo, Chem. Rev. 93 (1993) 1281e1306.
[19] ALOGPS 2.1 Software Available from: http://www.vcclab.org/lab/
alogps.
4.2.5. Statistical analyses
Data were expressed as means ꢂ SE, accompanied by the
[20] I.V. Tetko, V.Y.J. Tanchuk, J. Chem. Inf. Comput. Sci. 42 (2002)
1136e1145.
[21] P. Price, T.J. McMillan, Cancer Res. 50 (1990) 1392e1396.
number of experiments performed independently, and